Organization of DNA

1
Organization of DNA

• Viral DNA (and in some, RNA)
• Some circular, some linear
• Some double stranded, some single stranded
• Very small amount, packed very tightly
• Small size is an advantage
• Viruses use host cell enzymes, need few genes
• Bacterial DNA
• Usually single copy of double stranded
• Usually circular
• Eukaryotic DNA linear, in several pieces

2
DNA packaging
For example, the chromosome of E. coli is 1.2 mm
long, but must fit into a bacterium that is only
0.001 mm long!
http//www.expatica.com/xpat/xpatsite/www/upload_p
ix/surprised-face.jpg
3
Protein packaging of DNA

• Four proteins in E. coli
• Make up 10 of total protein of cell
• HU for wrapping FIS and IHF for bending HNS for
  compaction.
• Same function as histone proteins in eukaryotes.
• Positively charged proteins bind to negatively
  charged DNA.
• End result nucleoid, a region in the cytoplasm
  rich in DNA and protein comparable to a nucleus
  but without a membrane.

4
DNA of E. coli is supercoiled

• In addition to being packaged with proteins, the
  DNA of E. coli is supercoiled.
• Supercoiling. DNA could be relaxed or
  supercoiled. In Eubacteria, DNA is underwound
  (negatively supercoiled)
• Supercoiling carried out by topoisomerases.
• Example gyrase, that relieves stress during DNA
  replication.
• Two types, depending on whether 1 or two DNA
  strands are cut (and repaired) in the process.

5
Supercoiling
Top left relaxed DNA Bottom left
supercoiled. Bottom schematic of underwinding
DNA.
6
Packaging of E. coli DNA
Note arrows one shows where the DNA has been
nicked, relaxing the supercoiling. The other
points to a supercoiled region. That supercoiling
can be relaxed in ONE PLACE means that the DNA is
constrained in places.
7
The enslaved bacteria

• Mitochondria and chloroplasts thought to have
  originated as prokaryotic endosymbionts in early
  eukaryotes
• Carry out respiratory functions in membrane
• DNA is circular, ds DNA like in prokaryotes
• Self replicating
• Have their own ribosomes, similar to bacterial
• Organelle DNA discovered from mutations
• Some traits not determined by nuclear genes
• Inheritance via mother ovum has all the cytoplasm

8
Integration of organelles is thorough

• Mitochondria
• Replication requires nuclear genes
• Polymerases, initiation factors, respiratory
  proteins are multi-subunit proteins
• Several of the subunits for each are nuclear,
  others are mitochondrial
• Chloroplasts
• Multi-subunit enzymes jointly encoded
• Genes for RuBP carboxylase divided between
  nucleus and chloroplast

http//cellbio.utmb.edu/cellbio/mitoch2.htm
9
Polytene chromosomes
Occur in the salivary glands of various flies
during development. Condensed areas of DNA line
up, produce darkly staining bands. Useful for
mapping genes banding patterns are unique, and
in situ hybridization can be used to localize
genes on DNA
10
DNA packaging in eukaryotes

• Largest human chromosome is made of DNA which is
  82 mm long (over 3 inches)
• During metaphase, DNA is further compacted to
  about 10 µm long.
• Equivalent to winding 25 miles of spaghetti into
  a 16 foot canoe.
• DNA has to be well packaged to fit into the cell,
  to be compacted even more during mitosis
• still has to be accessible during interphase for
  use!
• Chromatin grainy appearing mixture of DNA and
  proteins in the nucelus

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Nucleosomes unit of packaging of eukaryotic DNA
DNA wrapped around histone proteins TWO each of
the proteins H2A, H2B, H3, and H4. Additionally,
H1 on outside helps hold DNA to structure.
12
About histones and arrangement

• Histones
• positively charged, to attach well to DNA
• conserved, very little difference among organisms
• How arrangement was determined
• DNA collected, treated briefly with nuclease to
  see how much DNA is protected by proteins
• Remove proteins, separate DNA pieces by size on
  gel
• 200 bp pieces of DNA produced
• treat more with nuclease, repeat analysis
• get 145 bp DNA pieces

13
Structure deduced

• the 145 bp of DNA are wrapped around the histone
  octet which is the core particle.
• 200 bp includes region covered by H1 which covers
  DNA as it enters, exits nucleosome.
• the rest of the DNA is linker DNA between.

14
Nucleosomes are wound up
Figure shows how beads on a string are further
wound up to produce a solenoid, the structure of
chromatin. During mitosis, this solenoid itself
coils further to make chromatids.
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Organization of DNA

• Does DNA packaging create problems?
• DNA wrapped tightly around histones
• DNA must be accessible for replication,
  transcription
• Modification of histones changes packing with DNA
• Acetylation acetylases added to histones.
• Phosphorylation phosphate groups added by
  kinases
• These groups decrease positive net positive
  charges, allow DNA freedom.
• Negative supercoiling helps too.

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Differences in DNA

• Heterochromatin vs. Euchromatin
• Heterochromatin is DNA which tends to be highly
  compacted and dark staining.
• Euchromatin is not so compacted or dark.
• The number of genes in heterochromatin is
  generally small relative to euchromatin.
• Heterochromatin lacks genes or they are inactive
• Much heterochromatin is found in certain
  structural parts of the chromosomes centromeres
  and telomeres. Also, much of Y chromosome.
• Move euchromatin to an area next to
  heterochromatin and it becomes heterochromatin
  position effect.

17
Chromosome structure
Arm
http//www.med.uiuc.edu/m1/genetics/images/webun1/
Chromosome.gif medic.med.uth.tmc.edu/.../
cellbio/hist-01.htm
18
Composition of DNA GC
There is always equal s of A and T, and G and C,
but the percentage of GC pairs and AT pairs
can be different among different organisms.
19
Measuring GC hyperchromic shift
As DNA melts, becomes SS, absorbs more UV at
260 nm. Because G-C pairs have 3 H-bonds instead
of two, DNA with more GC is more stable, melts
at higher temperature (blue).
20
Satellite DNA

• In prokaryotes, the GC base pairs is pretty
  much averaged out over the entire DNA not so
  with eukaryotes.
• Density gradient ultracentrifugation can also be
  used to determine GC.
• GC pairs are denser than AT, migrate to a lower
  location (greater density) in the gradient.
• Fragmented eukaryotic DNA showed something odd

21
Satellite DNA
When the DNA was analyzed, a portion has a lower
GC than the rest of the DNA, producing a
satellite band. How could a portion of DNA
have a different composition than the rest?
22
Repeated sequences

• If a section of DNA with a GC composition
  different from the rest of the DNA is repeated
  many times, DNA fragments from these regions of
  DNA would behave differently during the
  centrifugation.

23
Study of the Composition of DNA using DNA
renaturation kinetics

• Break DNA into random fragments.
• Denature with heat (melt).
• Cool, allow strands to find their complements and
  go from ss to ds again (anneal).
• Follow entire process using UV light absorption
  at 260 nm
• as DNA goes from ss to ds, Abs decreases.

24
Renaturation kinetics

• Kinetics study of the rate of change.
• Major Point 1 the more copies of the
  complementary strands there are, the less time
  they will take to
• find each other
• the more DNA,
• the faster the process.

In this fig., 2 different amounts of DNA from the
SAME organism.
25
Renaturation kinetics-2

• Major Point 2
• Given equal amounts
• (same mass) of DNA,
• the bigger the total genome
• of the organism, the slower
• the renaturation.
• If the genome is bigger, and the amounts of DNA
  used in the experiment are the same, the organism
  with the bigger genome will have fewer copies of
  the complementary fragments, so annealing will
  take longer (see point 1).

26
Understanding genome size
Imagine you have 20 playing cards. In one
instance, you have these 5 cards, another 5 cards
exactly the same, and 2 more sets of the Ace thru
10 but of diamonds. ltDeck 1gt In the second
instance, you have ace thru 5 of hearts and also
of diamonds. ltDeck 2gt
In which case will you match up pairs of hearts
and diamonds most quickly? The Deck 1 gets
matched up quicker.
http//www.skydiveelsinore.com/calendar/images/pla
ying-cards-spread.jpg
27
Cot curves Studying renaturation of DNA
The amount of DNA affects the rate at which DNA
fragments renature. To avoid the problem of
comparing samples with different amounts of DNA,
the change in ss DNA is graphed vs.the initial
DNA concentration (Co) x the time (t)
Cot Y-axis is the fraction or percent of the DNA
that is ss (experiment starts by denaturing the
DNA). X-axis is Cot which is a Log scale.
www.cas.muohio.edu/.../gene2000/ lect7/fig9p8c.jpg
28
Satellite DNA and Cot curves
When human DNA was analyzed this way, this was
the result
Remember the card deck experiment when there is
only one of each card in the deck, they take
longer to match up. So DNA that anneals quickly
must be in multiple copies
29
Cot curves and satellite DNA
Categories variable among different organisms.
Highly repetitive DNA, many complements, find
each other quickly. Single copy (unique sequence)
much slower.
http//www.ndsu.nodak.edu/instruct/mcclean/plsc431
/eukarychrom/cot2.gif
30
Types of DNA

• Unique, single copy typically 30-75 of DNA in
  most eukaryotes.
• Highly repetitive DNA 5-45 of DNA depending on
  species. In humans
• ALU family contains Alu I site. 300 bp long,
  appears 500,000 times, dispersed. 5 of DNA.
• SINEs short intersperesed elements
• transposable
• Alpha satellite DNA tandem repeats of 170 bp
  occur 5,000-15,000 times make up part of
  centromere. 6
• L1 family (in humans), example of LINEs
• Long interspersed elements
• transposable

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Middle or moderately repetitive DNA

• Moderately repetitive DNA
• Tandem or interspersed repeats
• VNTRs, good for DNA fingerprinting
• Variable number tandem repeats
• 15 100 bp long, between or within genes
• Dinucleotide repeats (CA)N, also good for
  forensic work
• in maize and yeasts transposons in large
  numbers.
• genes for rRNA, tRNA, ribosomal proteins,
  histones

32
All your DNA codes for proteins? Sorry, not
close

• Only 4 codes for proteins, in 30,000 genes
• 96 of DNA includes
• Introns, junk DNA within and around genes.
• Genes coding for rRNA and tRNA
• Junk DNA called repetitive sequences
• Pseudogenes have sequences that look like genes
  but are never expressed, dont work.
• We are related to everything else
• Our genes look like those from chimpanzees,
  bacteria.